The PLRC Grant Review committee has selected the awardees for the 2022-2023 Pilot and Feasibility Program grants. Our thanks to all who submitted proposals.
Congratulations to the following awardees of the 2022 PLRC Pilot and Feasibility Program grants:
Evan Delgado, PhD – Research Assistant Professor, Department of Pathology, Division of Medicine
“Understanding Immune Surveillance in Hepatocellular Carcinoma”
HCC is the 5th most common cause of cancer-related death in the United States with
an estimated 32,000 annual deaths. The standard of care for HCC as established by the IMBrave150 trial is a combination of the PD-L1 inhibitor, Atezolizumab, and an antiangiogenic, Bevacizumab. While this combination extends overall survival to 19.2 months in unresectable HCC, only 52% of patients survive to 18 months. Thus, clarifying underlying mechanisms regulating the immune response in HCC oncogenesis might strengthen immunotherapy efficacy.
There is a lack of progress in personalized therapeutics in HCC. The Wnt/ꞵ-catenin pathway has been approached as a target for precision medicine in HCC. We recently modeled HCCs with ꞵ-catenin mutations in mice up to 69% genetic similarity by co-expressing mutant ꞵ-catenin and the tyrosine kinase receptor MET (B+M). This model provides a clinically relevant tool for the investigation of personalized HCC therapeutics.
A recent study demonstrated HCCs with mutant ꞵ-catenin lack sufficient immune cell surveillance. These HCCs have poor cytotoxic T cell recruitment and respond poorly to checkpoint inhibitors. To better understand why, we compared the gene expression between Ctnnb1-knock out livers to those with Ctnnb1 mutations. A computational analysis of the differentially expressed genes indicated livers with Ctnnb1 mutations showed a pattern of reduced inflammation. A promoter analysis found at least 33 genes are regulated by the NFΚB pathway. Thus, activating NFΚB signaling might reverse immune suppression in CTNNB1 mutated HCCs. I aim to better understand ꞵ-catenin’s role in HCC immune suppression, and my central hypothesis is that HCCs with ꞵ-catenin mutations actively suppress both the innate and adaptive immune response. I will test this hypothesis in 2 specific aims: Aim 1 will activate NFKB signaling in B+M HCCs to test if we can induce a cytotoxic T cell response. Aim 2 will determine if MDSCs are playing an immunosuppressive role in ꞵ-catenin driven HCCs.
Overall, understanding ꞵ-catenin’s role in regulating immunosuppression in HCC will aid in the development of personalized immune therapies for HCC patients. I plan to pursue this work during my independent career and the PLRC provides an excellent environment for me to complete my designed studies. HCC is a significant public health burden, and I am committed to a career studying the disease.
With full support from my collaborators, Division Mentor, and Department Chair, I am confident that I will be able to complete the proposed research studies. My proposal builds on my current expertise and the protected time provided by the Pilot and Feasibility Award mechanism will enable me to gain experience in techniques and develop preliminary data that will inevitably support my independence.
Christopher Hughes, MD – Associate Professor, Department of Surgery, Division of Medicine
Flordeliza Villanueva, MD – Professor, Department of Cardiology, Division of Medicine
Raman Venkataramanan, PhD – Professor, Department of Pharmaceutical Sciences, Division of Pharmacy
“Sonothrombolysis Combines with Treprostinil Infusion During Machine Perfusion of Liver Allografts as Pre-Conditioning for Transplant”
Liver transplantation for end-stage liver disease is performed in about 8000 of patients annually in the United States and approximately 36,000 worldwide, with the dominant source of organs coming from deceased donors. Unfortunately, due to an insufficient number of useable donor livers, in the US alone about 14,000 patients remain waitlisted for transplant while more than 1,500 patients die awaiting transplantation each year. Further, as many as 20% of livers procured from deceased donors are unsuitable for transplantation due to ischemic injury that renders the organ non-viable.
Ex vivo liver machine perfusion is undergoing intense study as a potential method for improving liver allograft preservation prior to transplant. The potential to evaluate and/or treat an organ during normothermic machine perfusion (NMP) has increased utilization of extended-criteria donor organs initially declined for transplantation (1). Livers from donors after circulatory death (DCD) have received particular attention as this is the fastest growing subset of the deceased-donor organ supply, albeit one fraught with biliary and thrombotic vascular complications (2). Our goal is to define a process using mechanical and biochemical methods to minimize vascular thrombosis during NMP such that there is full and confluent reperfusion of a liver allograft upon transplantation, turning marginal, likely discarded, organs into ones for routine use.
In addition to the ischemia from direct warm ischemic time, the DCD liver exhibits oxidative stress and mitochondrial collapse during reperfusion, whether that be in the recipient or an NMP circuit (3). The peribiliary vascular plexus, a dense microvascular network supplying the oxygen-sensitive biliary tree, is particularly affected by thrombosis and ultimate ischemic injury. Clinically, this presents as ischemic cholangiopathy, a potentially fatal complication of liver transplantation. In one clinical study, 30% of DCD livers developed graft non-function or irreversible biliary duct injury, necessitating urgent re-transplantation (4). Our overarching goal is to define a process using mechanical and biochemical methods to minimize vascular thrombosis during NMP, such that there is full and confluent reperfusion of a liver allograft upon transplantation, turning marginal, likely discarded, organs into ones for routine use.
Sonothrombolysis (STL) involves the intravascular injection and microvascular circulation of ultrasound contrast microbubbles that are FDA-approved for echocardiographic imaging. Concurrently delivered ultrasound using acoustic parameters in the diagnostic imaging range causes microbubble cavitation, which in turn can mechanically disrupt intravascular thrombus. In pre-clinical and early clinical studies, sonothrombolysis has been shown to improve coronary epicardial and microvascular reperfusion in models of acute myocardial infarction. Drawing from this experience, we have injected ultrasound microbubbles into the perfusate of our novel ex vivo NMP system, onto which ischemic rat livers simulating DCD donors have been suspended, and delivered external ultrasound during perfusion. Our initial studies aimed at clearing the peribiliary plexus of dense microthrombi using sonothrombolysis showed improvement of treated livers by biochemical, histologic, and electron microscopic parameters.
To further augment the beneficial effects of sonothrombolysis, our goal with this study is to improve microbubble access to the microvasculature of the hepatic vasculature, including the peribiliary vascular plexus, by coupling sonothrombolysis and NMP with vasodilation. Treprostinil, a stable synthetic prostacyclin (PGI2), is a strong vasodilator which has been shown to partially ameliorate ischemia-reperfusion injury (IRI) in lung, kidney, and liver transplantation (5-7). Additionally, PGI2 has been shown to reduce platelet aggregation in vascular endothelia and apoptosis in rodent liver tissue (8). Several studies have found that Treprostinil also can reduce inflammation by inhibition of NF-kappa B activation (9). Treprostinil has a half-life of 4 hours, which allows for stable drug levels during ex vivo liver perfusion.
Edward Hurley – Assistant Professor, Department of Pediatrics, Division of Medicine
“Determining the Role of HSF1-Mediated Inhibition of Apoptosis in Hepatoblastoma”
Hepatoblastoma is the most common pediatric liver malignancy and while relatively rare, its incidence is increasing. Currently surgical resection with or without chemotherapy is the main treatment for the tumor though in severe cases children require liver transplantation. We have found hepatoblastoma tumors have increased gene expression levels of heat shock factor 1 (HSF1), a transcription factor which mediates cellular response to increased heat.
Our lab has discovered a method to make hepatoblastoma-like disease develop in mice. The resulting tumors are similar in appearance and genetically to aggressive hepatoblastoma in humans. This model can be used to test potential therapies for hepatoblastoma before human trials. We used this model to test how inhibiting HSF1 early in tumor development would impact cancer growth. We found less and smaller tumors when HSF1 was inhibited suggesting HSF1 is needed for aggressive tumor growth.
Broadly speaking, this research proposal will ask two questions. First, will inhibiting HSF1 after hepatoblastoma develops lead to decreased tumor growth from increased apoptosis in our mouse model? We will test both early disease and very advanced disease. We will use both a novel new genetic inhibitor of HSF1 and a chemical inhibitor that is currently available. We will also test cisplatin, which is one of the most commonly given chemotherapeutic agents for hepatoblastoma. Second, what transcriptional targets of HSF1 mediate apoptosis in hepatoblastoma? We will answer this question by performing chromatin immunoprecipitation followed by DNA sequencing (ChIP seq) on livers from lab’s mouse model.
This project will deeper knowledge into the role of HSF1 in hepatoblastoma development and growth and may lead to novel therapeutic agents to treat aggressive tumors that today are fatal or necessitate liver transplantation.
Pengfei Xu, PhD – Postdoctoral Research Associate, Department of Pathology
“PAPSS2-mediated Sulfation in Drug-induced Liver Toxicity”
Protein tyrosine sulfation is a common post-translational modification that affects protein-protein interactions and cellular signaling. The sulfoconjugation reactions depend on the unique and universal sulfate donor 3′- phosphoadenosine-5′-phosphosulfate (PAPS). The PAPS synthase 2 (PAPSS2) is the primary enzyme to synthesize PAPS in the liver. The goal of this study is to determine whether and how PAPSS2 plays a role in drug-induced hepatotoxicity.
Acetaminophen (APAP) overdose is a typical drug-induced liver injury and the leading cause of acute liver failure (ALF) in the United States. Oxidative stress and generation of reactive oxygen species are major pathogenic events in APAP overdose. As such, understanding APAP responsive stress response and its interventions will help to develop strategies to prevent and treat APAP hepatotoxicity. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of oxidative response through its transcriptional regulation of anti-oxidative genes. The activity of Nrf2 is positively regulated by p21. The p21 gene itself is a transcriptional target of p53, a tumor suppressor gene known to inhibit APAP hepatotoxicity. The transcriptional activity and stability of p53 is negatively regulated by its interaction with MDM2, an E3 ubiquitin ligase that targets p53 for ubiquitination and proteasomal degradation.
Our preliminary results showed that: 1) The hepatic expression of PAPSS2 is decreased in APAP-induced hepatotoxicity in mice and in human patients; 2) Hepatocytes are the major cellular source of PAPSS2 in the liver; 3) The hepatocyte specific Papss2 knockout mice (Papss2ΔHC) have been created; 4) Papss2ΔHC mice are protected from APAP-induced hepatotoxicity despite having a decreased APAP sulfation; 5) The hepatoprotective phenotype of Papss2ΔHC mice is accompanied by increased hepatic antioxidative capacity; 6) The increased hepatic antioxidative capacity is accompanied by the accumulation of p53 and induction of the p53 target gene p21, leading to enhanced activity of Nrf2; 7) Knock-down of p53 attenuates the hepatoprotective effect of Papss2ΔHC; and 8) p53 is a novel substrate of sulfation, and Papss2 ablation leads to decreased p53-MDM2 interaction.
Based on our preliminary data, we hypothesize that PAPSS2-facilitated protein sulfation plays an important in APAP hepatotoxicity. Specifically, we hypothesize that inhibition of p53 sulfoconjugation prevents APAP-induced oxidative liver injury by activating the p53-p21-Nrf2 axis. Mechanistically, p53 is a novel substrate of sulfation. Papss2 ablation or inhibition of sulfation leads to p53 protein accumulation by preventing p53 sulfation, which disrupts p53-MDM2 interaction and p53 ubiquitination, and increases p53 protein stability. We propose two specific aims to test our hypothesis: 1) To determine whether the hepatoprotective effect of Papss2ΔHC is p53-dependent; and 2) To determine the mechanism by which sulfoconjugation of p53 affects its protein stability and its activity in inhibiting APAP hepatotoxicity.
To our knowledge, this study represents the first attempt to systematically evaluate the role of PAPSS2 and protein sulfoconjugation in APAP hepatotoxicity. Our results suggest that inhibition of sulfation, especially p53 sulfation, may be explored for the clinical management of APAP overdose.